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Diff quik staining kit

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The Diff-Quik Staining Kit is a laboratory reagent used for the rapid staining of blood and other cellular samples. It provides a quick and reliable method for the differentiation and identification of various cell types within a sample. The kit consists of three solutions that are sequentially applied to the sample, enabling the visualization of cellular morphology and structures.

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Lab products found in correlation

Axio observer a1, by Zeiss (3 mentions) Biocoat matrigel invasion chamber, by BD (2 mentions) Matrigel invasion assay, by BD (1 mentions) Polyethylene terephthalate membrane, by Corning (1 mentions) Amersham ecl western blotting analysis system, by GE Healthcare (1 mentions) Propidium iodide, by Thermo Fisher Scientific (1 mentions) Duke standards, by Thermo Fisher Scientific (1 mentions) Elisa kit for tumor necrosis factor α, by BD (1 mentions) Pre cast 15 gels, by Bio-Rad (1 mentions) Immbilon pdvf membranes, by Merck Group (1 mentions) Halt protease inhibitor cocktail mix, by Thermo Fisher Scientific (1 mentions) Annexin 5 alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Transwell chamber, by BD (1 mentions) Oil immersion 100 objective, by Nikon (1 mentions)

10 protocols using diff quik staining kit

1

Transwell Migration Assay Protocol

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A transwell migration system utilizing 8.0 µm pore polyethylene terephthalate (PET, BD Falcon, MA, USA) inserts was used for the migration experiments as we previously described29 (link). Briefly, inserts were placed in 12-well notch plate, and 5 × 104 cells were suspended in DMEM supplemented with 1.0% serum and were seeded in the upper chamber, while DMEM supplemented with 10% serum was used as attractant in the lower chamber. Twenty four hours later, non-migrating cells were gently removed using cotton swab, while migrated cells adherent to the lower surface of the membrane were stained with Diff-Quik Staining Kit (Siemens Healthcare Diagnostics, USA). The number of migrated cells was counted in six different random fields using bright-field microscope (Axio Observer-A1, Carl Zeiss, Germany) at 5X magnification.
Elango R., Vishnubalaji R., Manikandan M., Binhamdan S.I., Siyal A.A., Alshawakir Y.A., Alfayez M., Aldahmash A, & Alajez N.M. (2019). Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo. Scientific Reports, 9, 13696.
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2

Matrigel-based Invasion Assay for Pancreatic Cancer

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Invasion was measured using a matrigel invasion assay (BD Biosciences). In brief, chemoattractant was placed in the wells of a 24-well plate, and pancreatic cancer cells (1 × 105) were plated on each matrigel-coated insert. Cells were then pretreated with and without the corresponding drugs and allowed to invade through the matrigel for 24 h toward the bottom chamber, which contained media and the chemoattractant, CCL25. The invading cells were fixed and stained using DiffQuik staining kit (Siemens; Newark, DE), and those in 5 random adjacent fields at 200× magnification were counted under light microscopy.
Lee S., Heinrich E.L., Li L., Lu J., Choi A.H., Levy R.A., Wagner J.E., Yip M.L., Vaidehi N, & Kim J. (2015). CCR9‐mediated signaling through β‐catenin and identification of a novel CCR9 antagonist. Molecular Oncology, 9(8), 1599-1611.
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3

Cell Migration Assay Protocol

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Cells (2 × 106 in 2 mL of serum free medium) were plated in the upper chambers of the wells of tissue culture plates containing polyethylene terephthalate membranes (pore size 8 μm, 6 cm in diameter; Corning Glass Work, Corning, NY, USA). The lower chamber contained 3 mL standard medium with 10% FBS. The cells on the lower side of the filter were stained using a Diff‐quik staining kit (Siemens, Munich, Germany) after incubation for 6 h at 37°C, and the number of cells was counted in four microscopic fields per well at a magnification of ×10. Data were collected from three independently performed experiments.
Kusumoto H., Shintani Y., Kanzaki R., Kawamura T., Funaki S., Minami M., Nagatomo I., Morii E, & Okumura M. (2017). Podocalyxin influences malignant potential by controlling epithelial–mesenchymal transition in lung adenocarcinoma. Cancer Science, 108(3), 528-535.
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4

Clonogenic Assay of Cancer Cell Response to Small Molecule Inhibitors

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The colony forming ability of cancer cells treated with the indicated small molecule inhibitor was determined using clonogenic assay as described before26 (link). Briefly, 4 × 104 cells were seeded in 2 ml culture medium in 12-well flat-bottom tissue culture plate, followed by subsequent serial dilution (1:1 to 1:32) in the presence of PTC-209 (5.0 μM), palbociclib (5.0 μM), or combination of PTC-209 and palbociclib (5.0 μM each) compared to DMSO vehicle control. The media was changed with fresh media supplemented with the appropriate inhibitor every 3–4 days. On day 10, the plates were washed and stained with Diff-Quik Staining Kit (Siemens Healthcare Diagnostics, USA), and were subsequently scanned and the number of colonies were observed under inverted microscope.
Elango R., Vishnubalaji R., Manikandan M., Binhamdan S.I., Siyal A.A., Alshawakir Y.A., Alfayez M., Aldahmash A, & Alajez N.M. (2019). Concurrent targeting of BMI1 and CDK4/6 abrogates tumor growth in vitro and in vivo. Scientific Reports, 9, 13696.
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5

Comprehensive Molecular Techniques Protocol

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The Diff-Quik staining kit was obtained from Siemens Healthcare Diagnostics Inc. (Newark, DE). Primary antibodies were bought from Abcam (Cambridge, MA) whereas the secondary antibodies were supplied in the kits purchased from ImmunoCruz (Santa Cruz, CA) for immunohistochemical staining and the Amersham ECL Western Blotting Analysis System (GE Healthcare). Ladd industries (Burlington, VT) supplied the Epon epoxy resin. Annexin V Alexa Fluor® 488 and propidium iodide were bought from Invitrogen (Carlsbad, CA). The ELISA kit for Tumor Necrosis Factor-α (TNFα was obtained from (BD Biosciences, San Jose, CA). Pre-cast 15% gels were bought from Bio-Rad (Hercules, CA) and Immbilon PDVF membranes from Millipore (Millipore, Billerica, MA). The Halt protease inhibitor cocktail mix was obtained from Thermo Scientific (Pittsburgh, PA). PlasticsOne (Roanoke, VA) supplied the cannulae for microinjection. Duke Standards (0.5, 1.0 and 1.5 µm) were obtained from Thermo Scientific. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Jeitner T.M., Kalogiannis M., Patrick P.A., Gomolin I., Palaia T., Ragolia L., Brand D, & Delikatny E.J. (2015). Inflaming the diseased brain: a role for tainted melanins. Biochimica et biophysica acta, 1852(5), 937-950.
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6

Clonogenic Assay for Transfected MDA-MB-231 Cells

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The colony forming ability of transfected MDA-MB-231 cells with miR-205-5p or hsa-miR-214-3p or pre-miR-negative control was assessed employing the clonogenic assay as we described before (31 (link)). In brief, transfected cells (4 × 104) were seeded in 2 ml of DMEM in 12-well tissue culture plate. Subsequently, cells were serially diluted (1:1–1:32). The media was replaced with fresh media twice a week. Ten days later when colonies were formed, plates were washed and subsequently were stained with Diff-Quik Staining Kit (Siemens Healthcare Diagnostics, USA). The plates were then scanned and the number of formed colonies was counted.
Elango R., Alsaleh K.A., Vishnubalaji R., Manikandan M., Ali A.M., Abd El-Aziz N., Altheyab A., Al-Rikabi A., Alfayez M., Aldahmash A, & Alajez N.M. (2020). MicroRNA Expression Profiling on Paired Primary and Lymph Node Metastatic Breast Cancer Revealed Distinct microRNA Profile Associated With LNM. Frontiers in Oncology, 10, 756.
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7

Assessing the Invasive Potential of HT1080 Cells

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The invasive potential of HT1080 cells was assessed using a BioCoat Matrigel invasion chamber (BD Biosciences). The suspension containing 2.5×104 HT1080 cells was plated in 24-well chambers dishes, and incubated for 22 h at 37°C. Invasive cells were fixed and stained using a Diff-Quik staining kit (Dade Behring, Inc., Siemens Healthcare GmbH). Invasive cells were counted, and invasion rate was calculated using the procedure reported previously (15 (link)).
Hasegawa K., Saga R., Takahashi R., Fukui R., Chiba M., Okumura K., Tsuruga E, & Hosokawa Y. (2020). 4-methylumbelliferone inhibits clonogenic potency by suppressing high molecular weight-hyaluronan in fibrosarcoma cells. Oncology Letters, 19(4), 2801-2808.
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8

Transwell Assay for Cell Migration

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The ability of the cells to migrate and invade was determined using a Transwell Chamber assay (BD Biosciences) per the manufacturer’s instructions. Cells were treated with CUDC-907 versus vehicle at specified concentrations for 24 hours, and then plated in the upper Transwell Chamber at a density of 50,000 cells/mL in 500 mL total volume of medium without FBS. In the bottom well, 750 μL of DMEM supplemented with 10% FBS was added to act as a chemoattractant. Cells were incubated at 37°C for 22 hours, at which point the cells that migrated/invaded were fixed and stained using the Diff Quik staining kit (Dade Behring). Cells were then photographed and analyzed using ImageJ software.
Kotian S., Zhang L., Boufraqech M., Gaskins K., Gara S.K., Quezado M., Nilubol N, & Kebebew E. (2017). Dual Inhibition of HDAC and Tyrosine Kinase Signaling Pathways with CUDC-907 Inhibits Thyroid Cancer Growth and Metastases. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(17), 5044-5054.
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9

Breast Cancer Invasion Assay with IL-6

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Cellular invasion of breast cancer cells was assessed using BD BioCoat™ Matrigel™ Invasion Chamber (Becton Dickinson Labware, Franklin Lakes, NJ, USA). MCF-7 cells at a density of 2.5 × 104 per well were seeded in the upper chamber with RPMI-1640 serum free media. The lower chambers were filled either with 500 μL of RPMI-1640 media supplemented with 3% FBS (control), or with different concentrations of IL-6 (10 ng/mL, 25 ng/mL and 50 ng/mL) in RPMI supplemented with 3% FBS. After 72 h of incubation the experiments were stopped and cells were fixed and stained as described before [26] (link). Non-invasive cells that remained in Matrigel or were attached to the upper side of the filter were removed with cotton swabs. Cells on the lower side of the filter, which invade in response to different concentrations of IL-6, were stained using the Diff-Quik staining kit (Dade-Behring, Inc., Englewood Cliffs, NJ, USA) and counted using light microscopy. Invasive cells were quantified by counting three random microscopic fields and expressed as number of invaded cells.
Ibrahim S.A., El-Ghonaimy E.A., Hassan H., Mahana N., Mahmoud M.A., El-Mamlouk T., El-Shinawi M, & Mohamed M.M. (2016). Hormonal-receptor positive breast cancer: IL-6 augments invasion and lymph node metastasis via stimulating cathepsin B expression. Journal of Advanced Research, 7(5), 661-670.
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10

Comprehensive Semen Analysis Protocol

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Semen analyses were performed manually according to World Health Organization guidelines (version V) [6 ]; after complete liquefaction of the samples. Semen volume, pH, sperm concentration, motility, and morphology were measured according to established laboratory protocol. To evaluate sperm morphology, the slides were stained with a Diff-Quik staining kit (Dade Behring AG, Düdingen, Switzerland) and strict criteria were applied to sperm viewed under a Nikon microscope with an oil immersion ×100 objective (Nikon Company, Tokyo Japan). Sperm samples were considered to be ‘normal’ when parameters met or exceeded the WHO reference values: concentration ≥15×106/mL, total motility ≥40%, and morphology ≥4% [7 (link)]. Samples with one or more abnormal semen parameters were excluded from this study.
Chan D.Y, & Li T.C. (2017). Comparison of the external physical damages between laser-assisted and mechanical immobilized human sperm using scanning electronic microscopy. PLoS ONE, 12(11), e0188504.
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